Antigen-mediated exocytosis in RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and and elevation in concentration of cytosol Ca(2+) ((Ca2+)). However, the relationship between production the intracellular Ca(2+) releasing agent, inositol 1,4,5-trisphosphate, and the increase in (Ca(2+)) has been established from studies with intact cells. It is shown that cells labeled with (3H)myoinositol and permeabilized with streptolysin O do release (3H)inositol 1,4,5-triphosphate upon stimulation with antigen or guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) at low (less than nM) concentrations of free Ca(2+). The response, however, is amplified by increasing free Ca(2+) to 1 MuM. As cytosol proteins and presumably inositol 1,4,5-trisphosphate 3'kinase leak from the permeabilized cells, conversion of the trisphosphate to inositol 1,3,4,5- tetrakisphosphate is reduced. As a consequence, (3H)inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells. Nevertheless, (3H)inositol 1,4-bisphosphate is the major product in permeabilized cells even when (3H)inositol 1,4,5-trisphosphate is trapped by the presence of excess (500 MuM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C and that these two lipids have rapid turnover times (less than 120 sec) ) at maximal rates of stimulation.